Anti t bet pe ebio4b10

Purified CD8⁺ T cells, splenocytes and thymocytes were first stained for surface antigens and then treated with Foxp3 staining buffer set according to the manufacturer's directions (eBioscience). Anti-Eomes AlexaFluor 647 or eFluor 660 (Dan11mag, 1/75), anti-T-bet PE (eBio4B10, 1/100) and anti-CD49d FITC or PE (R1-2, 1/50) antibodies were purchased from eBioscience. Anti-CD8 PercP (53–6.7, 1/50), anti-CXCR3 APC (CXCR3-173, 1/50), anti-CD4 Pe-Cy7 (RM4-5, 1/100), anti-CD62L PE (1/100) or V450 (1/50) (MEL-14), anti-Bcl2 PE (3F11, 1/25), anti-Ki67 FITC (B56, 1/25), anti-CD44 FITC or V450 (IM7, 1/50), anti-CD127 Pe-Cy7 (SB/199, 1/50), anti-CD122 FITC (TM-BETA1, 1/50), anti-NK1.1 FITC (PK136, 1/50), anti-CD90.2 Pe-Cy7 (53-2.1, 1/100) and anti-IFNγ APC or PB or PE (XMG1.21/50) were purchased from BD biosciences. Anti-CD3 Pe-Cy7 (2C11, 1/100) was purchased from Biolegend.
In some experiments, brefeldin A (5 μg ml⁻¹, Sigma) was added in samples for 3 h at 37 °C before intracytoplasmic staining. Blood samples were directly stained for surface antigens and then treated with FACS lysing buffer (BD biosciences) as described in the product data sheet. All samples were fixed with 1% paraformaldehyde in PBS prior to their processing using a Cyan flow cytometer (Dako Cytomation).

For intracellular cytokine staining after differentiation, cells were restimulated with phorbol myristate acetate (50 ng ml⁻¹) and ionomycin (1 μg ml⁻¹) in the presence of brefeldin A (5 μg ml⁻¹, both from Sigma) for 4 h, fixed with formaldehyde (2%) and stained with anti-IL-9-allophycocyanin (RM9A4, Biolegend) and anti-IFN-γ-phycoerythrin (PE) (XMG 1.2, eBioscience) mAbs. For intracellular staining of transcription factors after differentiation, cells were fixed with a Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Dilutent (eBioscience) and then stained with: anti-Foxp3-PE (FJK-16, eBioscience), anti-T-bet-PE (eBio4B10, eBioscience), anti-GATA-3-eF660 (TWAJ, eBioscience), anti-IRF4-AIFI647, (3E4, eBioscience) or anti-IRF1-PE (D5E4, Cell Signaling) mAbs. All antibodies were used in a 1:500 dilution. The cells were analysed by flow cytometry using a FACSCalibur or a FACSAriaIII (BD, Biosciences) with the DIVA software (BD, Biosciences) or the FlowJo Software (Tree Star).